Placenta 30 (2009) 823–834

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Placenta

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Current Topic Unearthing the Roles of Imprinted in the Placenta

F.F. Bressan a, T.H.C. De Bem a, F. Perecin a, F.L. Lopes b, C.E. Ambrosio a, F.V. Meirelles a, M.A. Miglino c,* a Department of Basic Sciences, Faculty of Animal Sciences and Food Engineering, University of Sa˜o Paulo, Pirassununga, Brazil b Department of Human Genetics, McGill University, Montreal, Canada c Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil article info abstract

Article history: Mammalian fetal survival and growth are dependent on a well-established and functional placenta. Accepted 22 July 2009 Although transient, the placenta is the first organ to be formed during pregnancy and is responsible for important functions during development, such as the control of metabolism and fetal nutrition, gas and Keywords: metabolite exchange, and endocrine control. Epigenetic marks and expression patterns in early Epigenetics development play an essential role in embryo and fetal development. Specifically, the epigenetic Genomic imprinting phenomenon known as genomic imprinting, represented by the non-equivalence of the paternal and Knockout maternal genome, may be one of the most important regulatory pathways involved in the development Placentation Transgenesis and function of the placenta in eutherian mammals. A lack of pattern or an imprecise pattern of genomic imprinting can lead to either embryonic losses or a disruption in fetal and placental development. Genetically modified animals present a powerful approach for revealing the interplay between gene expression and placental function in vivo and allow a single gene disruption to be analyzed, particularly focusing on its role in placenta function. In this paper, we review the recent transgenic strategies that have been successfully created in order to provide a better understanding of the epigenetic patterns of the placenta, with a special focus on imprinted genes. We summarize a number of phenotypes derived from the genetic manipulation of imprinted genes and other epigenetic modulators in an attempt to demonstrate that gene-targeting studies have contributed considerably to the knowledge of placentation and conceptus development. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Continuous research on placentation and the myriad mecha- nisms controlling this process is needed to clarify the embryonic– In mammals, embryo development and survival, as well as endometrial interactions, and the use of animal models has a successful pregnancy, are dependent on the establishment of contributed greatly to this study [7]. In particular, genetically a functional maternal–fetal interface. This connection is initiated modified animals have provided much of the knowledge on the during the primary contact of the embryo, followed by embryo genetic control of placental development [8]. In fact, the use of implantation, which is characterized by fetal trophoblast cell transgenic models has enabled the creation and analysis of gene invasion into the maternal endometrium, and it culminates with regulation assays; the discovery of new roles for genes in placen- the generation of the chorioallantoic placenta [reviewed by [1]]. tation; and, most importantly, it has contributed to our under- Together, these processes are referred to as placentation [2]. standing of developmental and perinatal pathologies in animals The phenomenon of genomic imprinting has been demon- and humans. In the present review, we address the epigenetic strated extensively to play a key role in fetal development and events involved in embryogenesis, focusing on imprinted genes placentation [3,4]. Although the majority of imprinted genes are and the knowledge generated by transgenic models as tools to expressed in extraembryonic tissues, there is little information increase our understanding of the roles that imprinted genes play available on the mechanisms by which such mono-allelic gene in placentation and early development. expression regulates placental growth, development and function [5,6]. 2. Epigenetics and development

The placenta is the first organ to be formed during pregnancy. It * Corresponding author. Tel.: þ55 11 30917690. is responsible for the establishment of vascular connections E-mail address: [email protected] (M.A. Miglino). between mother and conceptus and allows for the exchange of gas,

0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.07.007 824 F.F. Bressan et al. / Placenta 30 (2009) 823–834 nutrients and waste. This organ is involved in immune protection of www.mgu.har.mrc.ac.uk/imprinting, http://www.geneimprint.com, the fetus and also produces the hormones needed to support fetal http://igc.otago.ac.nz). In most genes, the imprinting status is development [9]. conserved between mouse and human [25] and in some genes the The creation of an appropriate maternal environment for fetal imprinted status is reported to be conserved also in other species, i.e., development depends on the proper functioning and development cattle [31–34]. As summarized in Table 1,imprintedgeneexpression of the trophoblast cells, which require the well-coordinated can be found in the placenta, the fetus, or both, independently of the expression of many transcription factors, cell cycle regulators, parental origin of the expressed allele, and may be widespread or growth factors, cytokines and surface receptors [reviewed by specific to certain cell types [4]. Although imprinted gene functions [10,11]]. Embryogenesis and placentation are particularly prone to are generally essential for the proper development and function of perturbations in gene expression because these processes depend the placenta, as well as for fetal growth [6],someofthesegeneshave on a complex cascade of events [12,13]. Any disruption to the well- not been reported to be related to development. It is important to orchestrated expression of these regulatory factors may lead to note, however, that imprinted genes can show spatial-temporal placental disorders, causing undesirable phenotypes or even expression [35]. Their expression window during development, precocious deaths in animals or humans [9]. therefore, may be narrow enough to cause the imprinted character- Following fertilization, a single-cell zygote forms a multicellular istic to be difficult to recognize. organism comprised of more than 200 different cell types [14,15]. The placenta is one of the most important sites of imprinted The development of lineage-specific cells begins with the differ- gene action [[36] reviewed by [37]]. Although placentation displays entiation of the trophoblast lineage and the inner cell mass [16]. species-specific variation [2], the genomic imprinting phenomenon This event depends on epigenetic modifications that control the is conserved amongst eutherian mammals, especially primates, expression of particular genes, allowing cells to develop and rodents and ruminants [6,38]. differentiate into specific cells and tissues [17]. According to the conflict hypothesis [39,40], paternally Epigenetics can be defined as the heritable changes in gene expressed genes enhance fetal growth, while maternally expressed expression that are not caused by the changes in DNA sequence genes suppress fetal growth. One evolutionary explanation for this [18]. The best studied epigenetic mechanisms are DNA methylation hypothesis would be that by restricting fetal growth, females can and histone post-translational modifications, which interact with have a longer reproductive lifespan, assuring their reproductive each other and also with regulatory and non-coding RNAs success. In contrast, having more numerous and stronger progeny is [reviewed by [19]]. advantageous for males. The conflict hypothesis achieved some The paternal genome is actively demethylated within a few confirmation through observations made with mouse genome hours of fertilization, while the maternal genome is demethylated manipulation. Androgenote mice, which contain only paternal passively during the first cleavages in a species-dependent manner. DNA, have poorly developed embryonic components but better This demethylation, however, spares imprinted genes [20], which developed extraembryonic tissues, whereas gynogenotes show the must be maintained throughout development without being opposite phenotype [41]. ‘‘de novo’’ reprogrammed during the pre-implantation stages [21]. It is important to note that both the accurate establishment of Imprinted genes are expressed selectively from either the genomic imprints and the correct maintenance of genomic paternal or maternal allele. This specialized form of gene regulation imprints during embryogenesis are essential for normal embry- is necessary for normal development [22,23], as discussed below. In onic/placental development [42]. Epimutations affecting paternally imprinted genes, the paternal allele is epigenetically imprints can arise during imprint erasure, which occurs when modified, preventing its transcription and leading to mono-allelic germ cells migrate to the gonads in pre-natal stages, during maternal expression [18,24]. The same happens to the maternally either the imprint establishment that takes place during game- imprinted genes, in which the paternal allele is solely expressed. togenesis or imprint maintenance throughout the life of the These selectively expressed genes are believed to have an impor- organism [43,44]. tant role in the allocation of maternal resources to fetal growth A clear example of epigenetic disturbance in development is the [25,26]. interference caused by assisted reproductive techniques (ARTs). Imprinted genes are found throughout the mammalian genome, These techniques likely interfere with imprint establishment though their occurrence is not random. These genes tend to be (manipulation of gametes) or imprint maintenance (manipulation found in clusters that contain DNA sequences that are rich in CpG of pre-implantation embryos; [43]). nucleotides. These specific regions, called imprinting control regions (ICRs), are characterized by epigenetic marks, mainly DNA 4. Imprinted genes control mammalian development methylation and histone modifications, which influence the binding affinity of transcription activators/suppressors and recruit Insulin-like growth factor 2 (Igf2) was one of the first imprinted chromatin remodeling enzymes to locally change the structure and genes to be discovered [45]. Igf2 and its receptor, Igf2r, are essential function of chromatin [27]. The existence of control regions during fetal–placental development [46]. While the former is suggests that genomic imprinting may be controlled not only at the a maternally imprinted gene that codes for a growth factor involved single gene level but at the level of the [28]. in fetal and placental growth in mice and humans, the latter is Epigenetic marks present in single parental copies of imprinted a maternally expressed gene in mice involved in Igf2 degradation. regions are responsible for differential gene expression. Interest- Although recent studies demonstrated that IGF2r is not imprinted ingly, the maintenance of imprinting has been recently inferred to in humans [47,48], the relationship between these genes brings depend more on repressive histone methylation than on DNA strength to the conflict theory [49,50]. methylation in the placenta [6,29]. Igf2, together with H19, which is an imprinted non-coding transcript, is located in a cluster of imprinted genes in mouse 3. Genomic imprinting and placental development chromosome 7, syntenic to human chromosome 11p15.5 [51,52].A region upstream of H19 regulates imprinted expression of both of Approximately 200 genes are imprinted in the mammalian these genes [53]. The establishment and maintenance of DNA genome [30]. More than 70 imprinted genes in mice and at least 50 in methylation in the Igf2/H19 DMR is acquired during spermato- humans have already been reported in the current literature (http:// genesis in the male germ cells; however, the DMR from the female F.F. Bressan et al. / Placenta 30 (2009) 823–834 825

Table 1 Imprinted gene expression reported in mouse development.

Gene Aliases Chromosome Preferentially Name References location imprinted allele

Gatm AT Central 2 Paternal L-Arginine:glycine amidinotransferase [140] (Extraembryonic tissues) Nnat Peg 5 Distal 2 Maternal Neuronatin [141] (Fetal brain) Nesp Distal 2 Paternal Neuroendocrine secretory [142,143] (Embryonic and extraembryonic tissues) Nespas Distal 2 Maternal Neuro endocrine secretory [143,144] (embryonic tissues) protein antisense Gnas Gs-alpha Distal 2 Maternal Guanine nucleotide binding protein, [145] (Embryonic tissues, alpha stimulating predicted by the embryonic lethality of null mutations) Gnasxl Distal 2 Maternal Guanine nucleotide binding protein, [142,146] (Embryonic tissues) alpha stimulating, ‘extra large’ Mcts2 Distal 2 Maternal Malignant T-cell amplified sequence 2 [147] (Embryonic tissues) H13 SPP Distal 2 Paternal Histocompatibility 13 [147] (Embryonic and extraembryonic tissues) Sfmbt2 Proximal 2 Maternal Scm-like with four mbt domains 2 [148] (Early embryos and extraembryonic tissues) Calcr Clr Proximal 6 Paternal Calcitonin receptor [149](Fetal brain) Mit1/Lb9 Proximal 6 Maternal Mest-linked imprinted transcript 1 [150] (Fetal brain, partially imprinted in other fetal tissues) Sgce e-SG Proximal 6 Maternal Sarcoglycan, epsilon [65,151] (Embryonic and extraembryonic tissues) Peg10 Edr, HB-1, Mar2, Proximal 6 Maternal Paternally expressed gene 10 [65] (Embryonic and MEF3L, Mart2, MyEF-3 extraembryonic tissues) Ppp1r9a Proximal 6 Paternal Neurabin [65,152] (Extraembryonic tissues) Pon3 Proximal 6 Paternal Paraoxonase 3 [65,152] (Extraembryonic tissues) Pon2 Proximal 6 Paternal Paraoxonase 2 [65,152] (Placenta-specific) Asb4 Proximal 6 Paternal Ankyrin repeat and suppressor [153] (Embryonic and of cytokine signalling extraembryonic tissues) Mest/Peg1 Proximal 6 Maternal Mesoderm specific transcript [154,155] (Embryonic and extraembryonic tissues) Copg2 Proximal 6 Paternal Coatomer protein complex subunit [150] (Embryonic tissues) gamma 2 Copg2as Proximal 6 Maternal Copg2 antisense [150] (Embryonic tissues) Klf4 Epfn, Klf14, epiprofin, BTEB5 Proximal 6 Paternal Kruppel-like factor 14 [156] (Embryonic and extraembryonic tissues) Nap1l5 Proximal 6 Maternal Nucleosome assembly protein 1-like 5 [157] (Embryonic tissues) Zfp264 Znf264 Proximal 7 Maternal Zinc-finger gene 264 [158] (Embryonic tissues) Zim3 Proximal 7 Paternal Zinc-finger gene 3 from imprinted domain [158] (Embryonic tissues) Kcnq1ot1 Kvlqt1as Distal 7 Maternal Kvlqt1 antisense [134,159] (Embryonic and extraembryonic tissues) Zim2 Proximal 7 Paternal Imprinted zinc-finger gene 3 [160] (Embryonic tissues) Zim1 Proximal 7 Paternal Imprinted zinc-finger gene 1 [161] (Embryonic tissues) Peg3 Pw1, End4, Gcap4, Zfp102 Proximal 7 Maternal Paternally expressed gene 3, probably Pw1 [161,162] (Embryonic tissues) Usp29 Ocat Proximal 7 Maternal Ubiquitin-specific processing protease 29 [163,164] (Mid-gestation embryos, fetal brain) Ube3a Hpve6a, E6-AP ubiquitin Central 7 Paternal E6-Ap ubiquitin protein ligase 3A [164] (Fetal brain) protein ligase Pwcr1 snoRNA MBII-85, Snord116 Central 7 Maternal Prader–Willi chromosome region 1 [165] (Embryonic tissues) Snrpn/Snurf Peg4, HCERN3 Central 7 Maternal Small nuclear ribonucleoprotein [166–168] (Embryonic and polypeptide N (Snrpn), extraembryonic tissues) Snrpn upstream reading frame (Snurf) Pec2 Central 7 Maternal Paternally expressed in the CNS 2 [164] (Fetal brain) Pec3 Central 7 Maternal Paternally expressed in the CNS 3 [164] (Fetal brain) Ndn Peg6 Central 7 Maternal necdin [164,169] (Fetal brain) Magel2 ns7, nM15, NDNL1, Mage-l2 Central 7 Maternal Melanoma antigen, family L, 2 [170] (Extraembryonic tissues and fetal brain) Mkrn3 Zfp127 Central 7 Maternal Ring zinc-finger encoding gene 127 [171,172] (Embryonic tissues) Zfp127as/Mkrnas Central 7 Maternal Ring zinc-finger encoding gene [173] (Pre-implantation embryo) 127 antisense Peg12/Frat3 Central 7 Maternal Frequently rearranged in advanced [174] (Embryonic tissues) T-cell lymphomas Inpp5f_v2 Distal 7 Maternal Inositol polyphosphate-5-phosphatase, [175] (Fetal brain) variant 2 Inpp5f_v3 Distal 7 Maternal Inositol polyphosphate-5-phosphatase, [147] (Fetal brain) variant 3 H19 Distal 7 Paternal [176,177] (Embryonic and extraembryonic tissues) Igf2 Mpr, M6pr, Peg2, Igf-2, Igf-II Distal 7 Maternal Insulin-like growth factor type 2 [45] (Embryonic and extraembryonic tissues) Ins2 Mody, Ins-2, InsII, Mody4, Distal 7 Maternal Insulin 2 [178,179] (Extraembryonic tissues) proinsulin, INS Ascl2/Mash2 Distal 7 Paternal Mus musculus achaete-scute homologue 2 [135] (Placenta-specific) (continued on next page) 826 F.F. Bressan et al. / Placenta 30 (2009) 823–834

Table 1 (continued )

Gene Aliases Chromosome Preferentially Name References location imprinted allele Tapa1/Cd81 Tspan28 Distal 7 Paternal cd 81 antigen [133] (Extraembryonic tissues) Tssc4 Distal 7 Paternal Tumor-suppressing subchromosomal [134,180] (Placenta-specific) transferable fragment 4 Kcnq1 Kvlqt1 Distal 7 Paternal Potassium voltage-gated channel, [134,180,181] (Embryonic and subfamily Q, member 1 extraembryonic tissues) Cdkn1c p57Kip2 Distal 7 Paternal Cyclin-dependent kinase inhibitor 1C [135,182] (Embryonic and extraembryonic tissues) Slc22a18 HET, ITM, Impt1, TSSC5, Orctl2, Distal 7 Paternal 22, member 18 [183,184] (Embryonic and Slc22a1l, Slc22a1, BWR1A extraembryonic tissues) Phlda2 Ipl, Tssc3 Distal 7 Paternal Pleckstrin homology-like domain, [185,186] (Weakly in embryonic, family A, member 2 (Phlda2), mainly in extraembryonic tissues) Imprinted in placenta and liver (Ipl) Nap1l4 Nap2 Distal 7 Paternal Nucleosome assembly protein 1-like 4 [181] (Mainly in placenta; however, reported not imprinted by [187]) Tnfrsf23 Tnfrh1 Distal 7 Maternal Tumor necrosis factor receptor [188] (Embryonic and superfamily, member 23 extraembryonic tissues) Obph1 Osbpl5 Distal 7 Paternal Oxysterol-binding protein 1 (Obph1), [181,189] (Placenta-specific) oxysterol binding protein-like 5 (Osbpl5) Plagl1 Lot1, Zac1 Proximal 10 Maternal Pleomorphic adenoma gene-like 1 [151] (Embryonic tissues) Dcn DC, PG40, PGII, Central 10 Paternal Decorin [153] (Placenta) PGS2, mDcn, DSPG2, SLRR1B Ddc Aadc Proximal 11 Maternal Dopa decarboxylase (Ddc); aromatic [190] (Embryonic heart) L-amino acid decarboxylase (Aadc) Grb10 Meg 1 Proximal 11 Paternal Growth factor receptor bound protein [191] (Embryonic tissues) U2afl1-rs1 SP2, 35 kDa, Irlgs2, Proximal 11 Maternal U2 small nuclear ribonucleoprotein [192] (Embryonic tissues) D11Ncvs75, auxiliary factor (U2AF), 35 kDa, U2afbp-rs, Zrsr1 related sequence 1 Mirg Meg9 Distal 12 Paternal miRNA containing gene [193] (Embryonic and extraembryonic tissues) Dlk1 FA1, ZOG, pG2, Distal 12 Maternal Delta-like 1 [194,195] (Embryonic and Peg9, SCP1, extraembryonic tissues) Ly107, pref-1 Gtl2 Meg 3 Distal 12 Maternal Gene trap locus 2 [195,196] (Embryonic and extraembryonic tissues) Rtl1 Mar, Mor1, Mart1, Distal 12 Maternal Retrotransposon-like 1 [196] (Embryonic and Peg11 extraembryonic tissues) Dio3 Distal 12 Maternal Deiodinase, iodothyronine type III [197] (Embryonic tissues and weakly imprinted in extraembryonic tissues) Antipeg11/Rtl1as Hosts several miRNAs Distal 12 Paternal Antisense to Rtl1/Peg11 [198] (Embryonic and extraembryonic tissues) Htr2a Htr2, Htr-2, 5-HT2A Distal 14 Paternal 5-Hydroxytryptamine [199] (Embryonic eye) receptor () receptor 2 A Kcnk9 Task3 Distal 15 Paternal Potassium channel, subfamily K, [200] (Embryonic tissues) member 9 Peg13 Distal 15 Maternal Paternally expressed 13 [157] (Embryonic and extraembryonic tissues) Slc238a4 Ata3, mATA3 Distal 15 Maternal Solute carrier family 38, [153] (Embryonic and member 4/amino extraembryonic tissues) acid transport system A3 Slc22a3 EMT, Oct3, Orct3, Slca22a3 Proximal 17 Paternal Solute carrier family 22 [201] (Placenta-specific) (organic cation transporter), member 3 Slc22a2 Oct2, Orct2 Proximal 17 Paternal Solute carrier family 22 [202] (Placenta-specific) (organic cation transporter), member 2 Igf2r CD222, CI-MPR, Mpr300, Proximal 17 Paternal Insulin-like growth factor type 2 receptor [203,204] (Embryonic and M6P/IGF2R extraembryonic tissues) Airn Air, Igf2ras Proximal 17 Maternal Insulin-like growth factor 2 [57,205] (Embryonic and receptor antisense RNA extraembryonic tissues) germline cell is protected against methylation by the zinc-finger successfully produced viable parthenogenetic offspring in mice by protein CTCF [52]. Such protection prevents interactions between correcting the Igf2/H19 dosage. In this experiment, one of the the Igf2 gene and enhancers located downstream of H19 in the maternal alleles was derived from a non-growing oocyte (ng), maternal allele, thus preventing Igf2 transcription. When CTCF does while the other was derived from a fully grown (fg) oocyte. The not bind to the paternal allele, on the other hand, Igf2 is expressed, process of imprinting in the maternal germline occurs at late stages and DNA is methylated within the H19 promoter region, resulting in of oogenesis. Therefore, ng oocytes are considered to be ‘‘imprint- H19 transcriptional silencing. The different methylation status of neutral’’, and both H19 and Igf2 genes are expressed [43,55,56].By the Igf2–H19 locus, therefore, guarantees the exclusive paternal Igf2 introducing a deletion in the H19 gene and its flanking regions in expression and maternal H19 expression [51]. the ng oocyte and consequently disrupting the imprinting of Igf2 The importance of the parental origin of Igf2/H19 genes was gene, the authors demonstrated both that parthenogenetic devel- elegantly demonstrated when Kono and collaborators (2004, [54]) opment to term could be achieved and also that the proper F.F. Bressan et al. / Placenta 30 (2009) 823–834 827 expression of Igf2/H19 likely drove modifications of other genes A recently discovered imprinted retrotransposon-derived gene, that allow parthenote survival. Peg10 [63], showed an essential function as an endogenous gene in The Igf2r cluster, which contains Slc22a2 and Slc22a3 genes, placental development [64]. Peg10 is highly conserved among a solute carrier family 22 that codifies imprinted genes, is also mammalian species [65], raising questions about its importance in regulated by methylation-sensitive elements. Unlike most imprin- mammalian evolution. Ono and collaborators [64] highlighted the ted genes, the methylated allele is expressed in this cluster. In this possibility that ancestral mammals may have developed placenta gene, the maternally methylated allele leads to paternal Igf2r from newly acquired retrotransposon-derived genes or by modifi- repression. The paternal non-methylated allele expresses a non- cation of endogenous genes present in oviparous animals millions coding RNA (ncRNA), called Airn (previously named Air), which is of years ago. The understanding of the physiological roles of Peg10 responsible for preventing paternal Igf2r expression [52,57]. and the other imprinted retrotransposon homologue Rtl1 is defi- Other important imprinted loci display the same behavior. The nitely important to improving our understanding of placental Gnas and Kcnq1 loci, for example, contain ncRNAs believed to evolution. contribute to genomic imprint control, i.e., Nespas/Gnas-as and Disrupting the normal regulation of imprinted genes is decisive Kcnq1ot1, respectively. Therefore, in addition to DNA methylation throughout gestation and post-natal life, often leading to lethal and post-translational histone modification, ncRNAs also control phenotypes in early development, as described in Table 2.Not imprinted gene expression [58]. surprisingly, these phenotypes are related to several human The mechanisms by which ncRNAs are responsible for the syndromes and disorders in post-natal life. epigenetic changes observed in these imprinted loci are still not The IGF2 gene, for example, is involved in Russell–Silver well characterized. Numerous ncRNAs are located in clusters syndrome (RSS), which is characterized by the loss of methylation regulated by ICRs [59]. In fact, each imprinted region expresses at in IGF2–H19 ICR, reduction in IGF2 expression, and biallelic least one ncRNA [58,60]. Although their function and mechanisms expression of H19, resulting in intrauterine and post-natal growth are not well understood, it is known that ncRNAs regulate retardation [66]. Beckwith–Wiedemann syndrome (BWS), on the imprinted clusters that recruit chromatin remodeling complexes to other hand, is characterized by the loss of IGF2 imprinting, causing nearby genomic regions. The expression of specific ncRNAs, i.e., biallelic overexpression and a lack of expression of H19, leading to long ncRNAs, is associated with the acquisition of genomic overgrowth of the fetus, among other symptoms. Both BWS and imprinting and the silencing of imprinting clusters [61,62]. RSS phenotypes include pronounced growth disorders [67].

Table 2 Imprinted genes knockout and their phenotypes.

Imprinted gene Mouse KO phenotype References Nesp Development without any obvious phenotype – behavior linked [206] Gnas Embryonic lethality. Heterozygous disruption is associated with significant early post-natal lethality. When maternal allele is disrupted [145,207] mice become obese. When paternal allele is disrupted, mice are hypermetabolic and thin Sgce Increased myoclonus and deficits in motor coordination and balance [208] Peg10 Growth retardation and early embryonic lethality due to incomplete placenta formation [64] Ppp1r9a Reduction in contextual fear memory, loss of hippocampal long-term potentiation [209] Mest/Peg1 Embryonic and placental growth retardation [210] Klf4 Neonatal lethality within 15 hours of birth, selective perturbation of late-stage differentiation structures in the epidermis [211] Kcnq1ot1 Reduction of 10–20% of weight [212] Peg3 Embryonic and placental growth retardation, impairment of normal maternal behavior [213] Ube3a Motor dysfunction, inducible seizures, context-dependent learning deficit [214] Pwcr1 Severe post-natal growth retardation, delayed sexual maturation, but fertile. Elevated level of anxiety or fear. Motor learning deficiency, [215] hyperphagie Snrpn Viable offspring, with no obvious phenotypic or histopathologic defects. However, KO of its IC leads to increase in neonatal mortality and [216] underweight newborns showing hypotonia Ndn Neonatal lethality and respiratory distress, underweight at birth [217,218] Magel2 Reduced viability at embryonic day 12.5. Offspring showing disregulation of sleep and food intake, growth retardation soon after birth [219] Peg12/Frat3 Viable, healthy and fertile. No obvious phenotype. Triple Frat knockout (Frat1, Frat2 and Frat3) shows the same normal phenotype [220] H19 Increase in placental weight, fetal overgrowth [221] Igf2 P0 and null mutants showed reduced placental growth, followed by fetal growth restriction. Phenotypes more severe in Igf2 null [26,46] mutants at later stages of gestation Ins2 Viable and fertile, without major metabolic disorders. Ins1 and Ins2 double homozygous knockout, however, were growth-retarded, [222,223] developed diabetes mellitus and died within 48 h Ascl2/Mash2 Death at 10 d post-coitum, placental failure [224] Tapa1/Cd81 Reduction of female fertility, increase in post-natal lethality [225] Kcnq1 Deafness, circular movement and repetitive falling. Gastric hyperplasia. Severe anatomic disruption of cochlear and vestibular end [226] organs. Phenotypes unrelated to BWS Cdkn1c Divergent phenotypes in offspring. Abnormal placental development (placentomegaly and trophoblast dysplasia), morphological [227–229] defects in neonates Phlda2 Placental overgrowth, consequent reduction of fetal-to-placental weight ratio [186] Plagl1 Intrauterine growth restriction, altered bone formation, increased neonatal lethality [230] Dcn Skin fragility, tumor development [231,232] Grb10 Embryo and placenta overgrowth [233] Dlk1 Pre- and post-natal growth retardation, eyelid and skeletal abnormalities, smaller litter size, increased neonatal mortality [234] Gtl2 Fetal and post-natal growth reduction [235] Rtl1 Placental abnormalities and functional deficiencies, pre- and post-natal growth retardation, placental growth retardation, increased [196] late-fetal or neonatal lethality Slc238a4 Placental and fetal growth restriction [236] Slc22a3 Impairment of neurotransmitters release. No obvious phenotypes, viable and fertile offspring [202] Slc22a2 No obvious phenotypes, viable and fertile offspring [237] Igf2r Lethality at birth, embryo overgrowth [238] Air Reduction in birth weight [57,239] 828 F.F. Bressan et al. / Placenta 30 (2009) 823–834

Abnormal imprinting patterns are also associated with neuro- context, the generation of in vivo gene function assays is vital for developmental disorders, such as Prader–Willi (PWS) and Angel- understanding the biological roles of developmental genes and man (AS) syndromes, which are associated with the loss of paternal their interactions with each other and with environmental stimuli. or maternal imprinting on chromosome 15q11–q13, respectively [reviewed by [14,23,68]]. 6. Transgenic strategies to study mammalian development

5. Imprinting alterations and implications Understanding the genetic control of fetal–maternal interac- tions has dramatically improved with the introduction of genome In humans, pregnancy losses are extremely common and not modifications in animal models. In fact, gene-targeting strategies completely understood. In fact, 25% of spontaneous abortions are the most widely accepted models used to provide reliable and remain unexplained [69]. The majority of these losses occur during accurate information on the mechanisms of implantation and the pre-implantation period, though after implantation, approxi- placentation, given their ability to provide definitive evidence for mately 15–20% of pregnancies are also lost spontaneously [70,71]. the in vivo function of a specific gene. In farm animals, embryonic mortality is also the major cause of Genes that are candidates to have a role in early development reproductive wastage, where a dysfunctional placenta accounts for can have their biological effects analyzed in vivo in one of the two 80% of this mortality [72,73]. ways: gain of function or loss of function studies. The first method ARTs have been widely used in an attempt to correct fertility is based on gene overexpression, achieved by the random inte- impairment in humans and animals and to provide a higher gration of a transgene into the genome or a targeted insertion of the reproductive efficiency in farm animals. In 2003, almost 4% of the transgene into a specific locus (a knock-in). On the contrary, the loss total number of human births in developed countries was esti- of function gene assay relies on the suppression of a gene function. mated to have been produced with in vitro procedures [74]. This Mainly, it is achieved by gene-trapping in ES cells or targeted gene scenario is not different for farm species. The last report of the IETS deletion (a knockout, KO). The first method, although relatively (International Embryo Transfer Society), released in 2006, inexpensive, has the significant limitation of being only effective for announced that in the previous year, nearly 266,000 bovine genes that are expressed in ES cells, whereas gene targeting can be embryos were produced in vitro and transferred worldwide. used for any gene, either permanently or in a conditional manner Despite its wide use, ARTs, such as IVF or cloning in animals, [reviewed by [94–96]]. increase the incidence of abnormalities in the morphology and The gain of function strategy is especially interesting for char- function of the placenta [75]. Hydroallantois, poor vascularization acterizing placental features that are not fully described. The and abnormal (mostly reduced but also enlarged) placentomes are transfer of transgenic embryos expressing a reporter gene, such as some of the most common pathological alterations [76–78]. green fluorescent protein (GFP) or the b-galactosidase enzyme Overall growth of the placenta and other particular structures (LacZ), to wild-type recipients enables the precise discrimination of (such as the labyrinthine trophoblast), as well as regulation of uterine and trophoblast contributions to placental defects [97]. The specific transporters and channels needed for nutrient supply to inverse is also valid when wild-type blastocysts are introduced into the fetus, are frequently regulated or affected by imprinted genes mutant uterine tract [71]. This technique has been used for several [reviewed by [25]]. purposes, such as elucidating trophoblast invasion in hemochorial Placental perturbations also lead to high birth weights and placentas [98], demonstrating the spatial-temporal pattern of reduced survival rates, a condition known in ruminants as large imprinted gene expression in embryos [99] or revealing the X offspring syndrome (LOS, [79,80]). This condition is reminiscent of inactivation mechanism [100,101]. the BWS in humans and is correlated with IGF2R imprinting KO mice model is another strategy that has greatly contributed disruption [81]. The incidence of placental failures is especially to the understanding of several diseases and different biological important in cloning by nuclear transfer because such failures processes [reviewed by [94]], usually revealing a gene role by represent the major cause of pregnancy failure in these animals comparing the knockout phenotype with that of wild-type mice. [76,82–85]. Placental abnormalities in cloned animals are evident For example, it has been used to uncover basic mechanisms of DNA and appear frequently even in gestations carried to term [86,87]. repair [102], cancer research [103], diabetes [104], behavioral Furthermore, the use of ARTs and their in vitro culture condi- analysis [105], and developmental related processes [106,107], tions changes the methylation and expression patterns of imprin- among several others. ted genes [81,88]. In laboratory animals, 5–10% of non-manipulated Despite differences between mice and human morphology and embryos undergoes abnormal methylation reprogramming and endocrine function, the mouse is the most popular model organism fails to develop. However, embryos derived from some kinds of for studying mammalian genomic imprinting and other processes manipulation, for example, superovulation and in vitro culture, in eutherian animals [108]. Great advantages of mice when undoubtedly present a higher rate of methylation and/or compared to other animals are the availability of maternal- or imprinting abnormalities when compared to non-manipulated paternal-only derived embryos and the characteristics of these embryos [89,90]. When nuclear transfer is considered, methylation animals, such as uniparental chromosomal duplications (UPD), patterns are also abnormal and highly variable between individuals high fertility, low costs to maintain feeding and housing facilities, [91,92]. and responsiveness to a range of assisted reproductive technologies Imprinted loci disruption has been observed in a number of [94,109]. Most importantly however, is the availability of a fully human developmental disorders and cancers [reviewed by [93]]. sequenced genome for this species [110] and the technology For example, a loss of imprinting (LOI) has been found in patients available for the manipulation of embryonic stem cells, allowing with PWS (at a frequency of approximately 1%), patients with AS (at the use of these cells for the production of genetically altered a frequency of 3%), patients with BWS (50% of patients), and nearly offspring [111,112]. 50% of the transient neonatal diabetes mellitus [reviewed by The generation of KO mice relies on several in vitro procedures [44,67]]. that, although specific, are technically simple to perform. The first The observation that epigenetic abnormalities are present in step consists of the design and construction of the desired vector. normal or manipulated pregnancies has made the animal model Circular sections of bacterial DNA (plasmids) are frequently used to suitable for a more profound study of these perturbations. In this manipulate the genome of embryonic stem cells by introducing F.F. Bressan et al. / Placenta 30 (2009) 823–834 829 a DNA sequence flanked by homologous sequences into the gene to Five main mammalian DNA methyltransferases (Dnmt) have been be inactivated [113]. Reporter genes, as well as antibiotic resistance characterized and are related to the establishment and mainte- genes, are introduced into the center of the target gene, causing nance of genomic imprinting: Dnmt1, Dnmt1o, Dnmt3a, Dnmt3b interference with expression and also allowing for the positive and Dnmt3L [124]. Dnmt1 and the oocyte isoform Dnmt1o are selection of the transgene in the cell genome [114]. responsible for the maintenance of the imprinted methylation Homologous recombination of plasmid and DNA sequences is patterns [125,126], Dnmt3a and Dnmt3b are required for de novo obtained with a very low and variable efficiency rate [115]. Nor- methylation and are essential for paternal and maternal methyl- mally, it consists of the recombination of similar chromosome ation imprints during germline development [127]. Most recent sections derived from each parent [116]. Gene-targeting technolo- studies have shown that Dnmt3-like (Dnmt3L) cooperates with gies exploit this characteristic by recombining transgenes con- Dnmt3a and is necessary for the establishment of genomic taining a disrupted gene with a similar DNA sequence, leading to imprinting during gametogenesis [128–130]. By constructing KO targeted gene disruption. mice models, it was possible to show that these methyl- Successfully modified embryonic stem cells are injected in pre- transferases are indispensable for embryogenesis, as summarized implantation blastocoels, contributing to the tissues of the devel- in Table 3. oping animal, including the germline [117,118]. Embryonic and Similar to DNA methylation, histone modifications, mainly adult tissues are composed of transgenic and non-transgenic cells acetylation and methylation, also influence gene expression called chimeras. Once these embryonic stem cells are integrated [131,132]. In contrast to embryo formation, placentation seems to into germ cells, the newly inserted gene alteration may be passed be more dependent on repressive histone methylation than DNA on to the next generations. As a result, the chimeras produced are methylation, as stated earlier. Some imprints in extraembryonic able to generate mouse strains that are heterozygous for the altered tissues directly correlated with histone H3 repressive methylation genes, and, most importantly, homozygous offspring can be but not with DNA methylation [133,134]. In placenta, several genes obtained by planned matings [reviewed by [94]]. maintain imprinting status in the absence of Dnmt1 [135,136]. These genes probably have their DNA-methylated allele enriched 7. Developmental studies based on knockout models with histone H3-lysine-9 methylation, together with other histone lysine methylation. Using KO models, the histone methyltransfer- Transgenic approaches in mice have provided reliable means of ase (HMT) G9a was shown to contribute to the allelic repression of investigating complex biological phenomena or diseases by genes that are imprinted only in the trophoblast. The dependence allowing gene products to be expressed in a controlled manner in of histone post-translational modification in the parental origin- a whole organism where the majority of the genes have a human specific expression probably prevents imprint erasure during the counterpart [119,120]. genome-wide demethylation wave that occurs after fertilization Indeed, an International Mouse Knockout Consortium [29,136]. composed of four groups, the Knockout Mouse Project (KOMP, KO studies of other HMTs or histone deacetylases (HDACs) have http://knockoutmouse.or), the European Conditional Mouse shown that deletions of its encoding genes (i.e., HMTs Eset and G9a Mutagenesis Program (EUCOMM, http://www.eucomm.org), the and Polycomb-group genes Ezh2 and Suz12) lead to embryonic North American Conditional Mouse Mutagenesis Program (Nor- lethality [131,137–139]. The mechanisms by which histone modi- COMM, http://norcomm.phenogenomics.ca/index.htm), and the fiers regulate the maintenance of differentially allelic chromatin Texas Institute for Genomic Medicine (TIGM, http://tigm.org), was organization in imprints require further investigation. created in 2007 to obtain a mutation of all protein-encoding genes From more than 70 imprinted genes in which expression was in the mouse using a combination of gene-targeting and gene- already reported in developmental stages, roughly half have been trapping strategies [96,121]. analyzed through KO studies, which are summarized in Table 2. The Regarding developmental process, mouse mutants have been phenotypes observed in the KOs ranged from increased embryonic created for the broad study of gene expression and developmental or post-natal lethality (i.e., Gnas, Peg10, Klf4, Ascl2, Tapa1/CD81)to interactions not only throughout the peri-implantation and gesta- no obvious phenotypes (i.e., Nesp, Peg12/Frat3, Slc22a2). Most tion periods [reviewed by [1,9]] but also for different stages of phenotypes evaluated by KO experiments confirm the preferential reproduction [reviewed by [107,122]]. allelic gene expression and its importance for fetoplacental growth. KO models have been used for more than a decade to investi- For example, Table 2 shows that the deletion of the paternally gate gene function, including the role of certain genes for epige- expressed genes Peg10, Mest/Peg1, Peg3, Igf2, Dlk1, Gtl2, Rtl1 and netic patterning and embryogenesis. Trasler and collaborators in others suppresses growth, whereas the deletion of the maternally 1996 [123] showed that DNA methyltransferase (Dnmt/)KO expressed genes H19, Grb10, Igf2r and others increases fetopla- embryos failed to develop past the 25-somite stage and were cental growth. Some mutations, although apparently unrelated to developmentally delayed and asynchronous. The authors nutrition allocation and fetal growth, are essential for fetal devel- concluded that DNA methylation is vital for embryo development. opment, i.e., the deletion of Ube3a, Sgce, Ppp1r9a and Pwcr1, among

Table 3 DNA and histone methyltransferases knockout consequences.

Methyltransferases Knockout consequences References Dnmt1 Embryonic extensive demethylation [240] Dnmt1o Embryos from Dnmt1o/ females lose half of their imprints during one cell cycle [126] Dnmt3a Apparently normal at birth, increased lethality at about 4 weeks of age, presenting runted phenotype [127,241] Dnmt3b Embryonic lethality probably due to multiple developmental defects [241] Dnmt3a and Dnmt3b Impaired de novo methylation. Embryonic lethality before 11.5 dpc [241,242] Dnmt3L Null mutations reveal disruption of maternal methylation imprints. Heterozygous [128–130] progeny of homozygous females fail to develop beyond 10.5 dpc due to abnormal development of extraembryonic structures HMT G9a Decrease in H3-K9 methylation in placenta, embryonic lethality before/at 10 dpc [29,137] 830 F.F. Bressan et al. / Placenta 30 (2009) 823–834 others, and mainly result in impairments related to the nervous [10] Hemberger M, Cross JC. Genes governing placental development. 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